Running title: VQ29 regulates seedling de

Plant Physiology Preview. Published on February 25, 2014, as DOI:10.1104/pp.113.234492
Running title: VQ29 regulates seedling de-etiolation
Research area: Signaling and Response
Corresponding author:
Rongcheng Lin, 86-10-62836905, e-mail: [email protected]
Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences,
Beijing 100093, China
One-sentence summary: A new transcription regulator VQ29 interacts with the
transcription factor PIF1 to modulate hypocotyl cell growth in response to light.
1
Copyright 2014 by the American Society of Plant Biologists
Arabidopsis VQ-motif-containing Protein 29 Represses Seedling
De-etiolation by Interacting with PIF1
Yunliang Li,a,b Yanjun Jing,a Junjiao Li, a,c Gang Xu, a,b Rongcheng Lina,1
a
Key Laboratory of Photobiology, Institute of Botany, the Chinese Academy of
Sciences, Beijing 100093, China
b
University of the Chinese Academy of Sciences, Beijing 100049, China
c
College of Life Sciences, Capital Normal University, Beijing 100048, China
1
Corresponding author, e-mail: [email protected]
The author responsible for distribution of material integral to the findings presented in
this article in accordance with the policy described in the instructions for authors
(www.plantphysiol.org) is Rongcheng Lin ([email protected]).
2
Footnotes: This work was supported by grants from the National Natural Science
，
Foundation of China (31170221 31325002), and the Ministry of Agriculture of China
(2011ZX08009-003) to R.L.
3
ABSTRACT
Seedling de-etiolation, a critical process in early plant development, is regulated by an
intricate transcriptional network. Here, we identified VQ motif-containing protein 29
(VQ29) as a novel regulator of the photomorphogenic response in Arabidopsis
thaliana. We showed that 29 of the 34 VQ proteins present in Arabidopsis exhibit
transcriptional activity in plant cells, and that mutations in the VQ motif affect the
transcriptional activity of VQ29. We then functionally characterized VQ29 and
showed that the hypocotyl growth of plants overexpressing VQ29 is hyposensitive to
far-red and low intensity white light, whereas a vq29 loss-of-function mutant exhibits
decreased hypocotyl elongation under low intensity of far-red light or white light.
Consistent
with
this,
VQ29
expression
is
repressed
by
light
in
a
phytochrome-dependent manner. Intriguingly, our yeast two-hybrid, bimolecular
fluorescence complementation and co-immunoprecipitation assays showed that VQ29
physically interacts with PHYTOCHROME-INTERACTING FACTOR1 (PIF1). We
then showed that VQ29 and PIF1 directly bind to the promoter of a cell
elongation-related
gene,
XYLOGLUCAN
ENDOTRANSGLYCOSYLASE7,
and
co-activate its expression. Furthermore, the vq29 pif1 double mutant has shorter
hypocotyls than either of the corresponding single mutants. Therefore, our study
reveals that VQ29 is a negative transcriptional regulator of light-mediated inhibition
of hypocotyl elongation that likely promotes the transcriptional activity of PIF1
during early seedling development.
4
INTRODUCTION
Light is an important environmental signal that affects plant growth and development
throughout its life cycle, directing processes such as seed germination, seedling
de-etiolation, phototropism, circadian rhythms, shade avoidance, and flowering timing.
Dark-grown seedlings, which adopt a developmental program known as etiolation or
skotomorphogenesis, exhibit elongated hypocotyls and closed cotyledons with apical
hooks. Upon light irradiation, seedlings undergo de-etiolation or photomorphogenesis,
which slows hypocotyl growth and causes cotyledons to expand and chloroplasts and
chlorophylls to develop (von Arnim and Deng, 1996).
Intensive research has revealed the main signaling pathway governing
photomorphogenesis (Chen et al., 2004, Lau and Deng, 2010, Arsovski et al., 2012).
To initiate the light responses, plants rely on a set of photoreceptors, including the
red/far-red light-absorbing phytochromes (phys) and the blue/UV-A light-absorbing
cryptochromes (crys). Activation of photoreceptors transmits signals to key
downstream
negative
factors,
such
as
COP1
(CONSTITUTIVE
PHOTOMORPHOGENIC 1) and members of the PIF (PHYTOCHROME
INTERACTING FACTOR) protein family. COP1 is a RING-type E3 ubiquitin ligase
that targets photomorphogenesis-promoting factors, such as HY5 (ELONGATED
HYPOCOTYL 5) and HFR1 (LONG HYPOCOTYL IN FAR-RED 1), for 26S
proteasome-mediated degradation, which desensitizes the light pathway initiated by
both phys and crys (Wei and Deng, 1996, Lau and Deng, 2012). PIFs encode a group
of basic helix-loop-helix (bHLH) transcription factors (TFs) that are phosphorylated
and degraded in a phy-dependent manner in light. PIF proteins (including PIF1, 3, 4,
and 5) play redundant roles in directly regulating gene expression and repressing
photomorphogenic responses (Leivar et al., 2008, Shin et al., 2009, Leivar and Quail,
2010).
Genetic and molecular studies in Arabidopsis thaliana have identified another
series of signaling components that is involved in either negatively or positively
regulating the light pathway (Jiao et al., 2007). Notably, many of the identified
5
effectors are nuclear-localized TFs, such as FHY3 and FAR1 (transposase-derived
TFs), bZIP16 (bZIP TFs), HFR1 and MYC2 (bHLH TFs), LAF1 (a MYB TF), and
STH2 and LZF1 (B-box-containing TFs) (Hudson et al., 1999, Wang and Deng, 2002,
Ballesteros et al., 2001, Hsieh et al., 2012, Datta et al., 2007, Chang et al., 2011,
Yadav et al., 2005). Moreover, genes encoding F-box proteins (such as EID1 and
AFR), kinases (e.g., NDPK2), and phosphatases (such as PP7) were also isolated and
shown to mediate light signaling (Dieterle et al., 2001, Harmon and Kay, 2003, Choi
et al., 1999, Møller et al., 2003). However, the regulatory mechanisms underlying the
actions of these factors are not well understood. In addition, we recently showed that a
chromatin remodeling factor, EPP1 (enhanced photomorphogenesis 1) / PICKLE,
interacts with HY5 to fine-tune light signaling by modulating H3k27me3 levels on
cell elongation-related genes (Jing et al., 2013). Even though a wide range of light
signaling intermediates have been identified and extensively studied, a complete
picture of the light signaling pathway has yet to emerge.
A group of genes encoding proteins containing a unique and conserved
FxxxVQxxTG motif (termed the VQ motif) was recently identified (Xie et al., 2010,
Cheng et al., 2012). These proteins include 34 members in Arabidopsis and are
designated as VQ motif-containing proteins (VQ). The function of a few members of
this family has been characterized to date. For example, sigma factor binding protein
1 (SIB1/VQ23), its close homolog SIB2 (VQ16), and MAP kinase 4 substrate 1
(MKS1/VQ21) are required for the plant defense response (Narusaka et al., 2008, Xie
et al., 2010, Lai et al., 2011, Andereasson et al., 2005). In addition, IKU1 (VQ14) is a
regulator of endosperm growth and seed size (Wang et al., 2010), while CaMBP25
(VQ15) and VQ9 function as negative effectors of osmotic and salinity stress
tolerance, respectively (Perruc et al., 2004, Hu et al., 2013). It is anticipated that this
family responds to various environmental signals and plays diverse roles in plant
defense, growth, and development. However, the functions and regulatory
mechanisms of most VQ family members remain unknown.
In this study, we demonstrate that the VQ family of proteins largely possesses
transcriptional activities. Furthermore, we characterized the regulation and function of
6
VQ29 in detail. We show that VQ29 expression is down-regulated by light.
Overexpression of VQ29 results in hyposensitivity of hypocotyl growth to far-red and
low light conditions, whereas the vq29 loss-of-function mutant exhibits decreased
hypocotyl elongation under low intensity of far-red and white light, during seedling
de-etiolation. We also demonstrate that VQ29 physically interacts with PIF1 and that
these proteins cooperatively activate the expression of downstream genes. Our study
identifies a novel factor in photomorphogenesis and provides insight into the roles of
VQ family proteins in regulating diverse plant growth and developmental processes.
7
RESULTS
Analysis of VQ genes from Arabidopsis, rice, and moss
Previous studies documented that the VQ gene family is found only in plants and it
was systematically studied in Arabidopsis thaliana (Xie et al., 2010, Cheng et al.,
2012). To gain insight into the evolution of this family, we searched the GenBank
database for sequences of the VQ genes from rice (Oryza sativa) and moss
(Physcomitrella patens), which represent monocot and lower plants, respectively.
Whereas the model dicot plant Arabidopsis has 34 VQ members, rice and moss have
39 and 25, respectively. Interestingly, Arabidopsis and rice VQ proteins are relatively
small, with about 85% and 92% of the proteins containing fewer than 300 amino acid
residues, respectively. In contrast, 68% of the moss VQ proteins are longer than 300
amino acids (Supplemental Figure S1A). We further found that most VQ genes in
higher plants (30 in Arabidopsis and 37 in rice) do not contain an intron. However, in
moss, only 7 VQ genes do not have intron, whereas 5 VQs have one intron and 13
genes possess two or more introns (Supplemental Figure S1B). These results suggest
that VQ genes tend to be intronless and encode relatively small proteins in higher
plants.
VQ proteins exhibit transcriptional activity
To investigate whether the VQ proteins are involved in transcriptional regulation, we
attempted to isolate the open reading frame (ORF) of each VQ gene in Arabidopsis
Columbia (Col) plants using reverse transcription polymerase chain reaction (RT-PCR,
for VQ2) or PCR for other intronless VQ genes. The PCR primers were designed
according to the available cDNA sequence information or the predicted sequences.
The ORFs of all VQ genes were amplified and cloned into the pEASY vector and
verified by sequencing. We then subcloned the VQ genes in-frame with the GAL4
DNA-binding domain (GBD) and under the control of the cauliflower mosaic virus
(CaMV) 35S promoter in the pSAT-GAL4DB vector (Jing et al., 2013). The construct
was co-transformed with a luciferase reporter gene (LUC), driven by the 35S minimal
8
promoter and fused in-frame to a GAL4 binding sequence, into Arabidopsis
mesophyll protoplasts (Figure 1A). We found that GBD fusion proteins with VQ14,
VQ5, VQ15, VQ16, VQ9, VQ23, VQ3, VQ24, VQ34, VQ17, VQ32, or VQ30
drastically activated LUC reporter expression compared with GBD alone. The VQ26,
VQ12, VQ18, VQ28, and VQ6 fusions promoted LUC expression to a lesser extent
(Figure 1B). However, GBD fusions with VQ19, VQ31, VQ4, VQ8, VQ13, VQ33,
VQ11, VQ29, VQ7, VQ2, VQ21, or VQ20, remarkably repressed the expression of
the LUC reporter (Figure 1C). We also observed that GBD fusions with VQ22, VQ1,
VQ27, VQ25, and VQ10 did not affect the transcription of LUC (Figure 1B, C). These
data indicate that most members of the VQ family possess either transcriptional
activation or repression activities in this heterologous gene reporter system. In this
study, we focused on VQ29 (At4g37710) because it is involved in seedling
de-etiolation response (see below in detail).
Mutation in the VQ motif affects transcriptional activity
Since VQ family members contain only the VQ motif and possess activation or
repression activity, we asked whether the VQ motif is required for transcriptional
activity. We then altered conserved amino acids in the VQ motif of VQ29, where the
very hydrophobic residue valine (V) was changed into a less hydrophobic residue
alanine (A) or the hydrophilic residue aspartic acid (D), and the hydrophilic residue
glutamine (Q) was mutagenized into the hydrophobic residue leucine (L). As shown
in Figure 1D, single mutation of VQ29(V70A) or VQ29(V70D) abolished the
repressive activity of VQ29, and greatly activated LUC reporter gene expression,
whereas mutation in VQ29(Q71L) did not affect the activity. Furthermore, double
mutations in VQ29(V70D, Q71L) led to a significant induction of LUC to a lesser
extent than VQ29(V70D) (Figure 1D). It should be noted that two point mutations
(V70D and Q71L) as tested, did not affect the levels of the VQ29 protein
(Supplemental Figure S2). Taken together, these results suggest that the VQ motif is
likely involved in mediating the transcriptional activity of VQ proteins.
9
Overexpression of VQ29 reduces the hypocotyl growth response under far-red
and low intensity of white light conditions
To elucidate the biological function of VQ29, we obtained two T-DNA insertion lines
of VQ29, Salk_061586 and Salk_061438. PCR genotyping and sequencing studies
revealed that the T-DNA is inserted in the promoter region 136 base pairs upstream of
the ATG start codon of VQ29 in both mutants. These mutants thus represent the same
allele (Figure 2A). They had been backcrossed five times with the Col wild type and
thus the potential background mutations were largely eliminated. Quantitative
RT-PCR analysis showed that the VQ29 transcripts were barely detectable in the
mutant (hereafter referred to as vq29-1), suggesting that it is a null allele (Figure 2B).
We also generated transgenic plants over-expressing VQ29, fused with a MYC tag and
driven by the CaMV 35S promoter (Pro35S:Myc-VQ29, VQ29-OE). Over forty
transgenic lines were obtained, two of which (lines #10 and #14) were further studied
in the following experiments. Immunoblotting analysis using the MYC antibody
showed that both lines accumulated Myc-VQ29 fusion protein, with line #10 having a
higher level of expression than line #14 (Figure 2C).
We then tested the hypocotyl growth response of the vq29-1 mutant and VQ29
overexpression lines under various light conditions. The hypocotyl length of vq29-1
was slightly but significantly shorter than the wild type under low intensity of white
light (less than 20 µmol m-2 s-1) or low intensity of far-red light (less than 12 µmol m-2
s-1), but was indistinguishable from that of the wild type in red or blue light conditions
with multiple fluence rates tested (Figure 2D, 2E, Supplemental Figure S3). However,
the hypocotyls of VQ29-OE transgenic seedlings were significantly longer than those
of wild-type plants under far-red conditions, with line #10 exhibiting the stronger
phenotype, consistent with its higher VQ29 level (Figure 2C-E, Supplemental Figure
S3). The hypocotyls of overexpression line #10 were also longer than the wild type
under low white light conditions, but not in darkness or in red or blue light conditions
(Figure 2E, Supplemental Figure S3). It has been documented that sucrose promotes
seedling growth (Stewart et al., 2011). Even without sucrose supplement in the MS
media, vq29-1 exhibited reduced hypocotyl elongation under low white light and
10
different intensities of far-red light, and the hypocotyl length of VQ29-OE plants (line
#10) was longer than the wild type under these conditions (Supplemental Figure S4).
Moreover, the expression of two light-responsive genes involved in cell elongation,
PHYTOCHROME INTERACTING FACTOR 3-LIKE1 (PIL1) and XYLOGLUCAN
ENDOTRANSGLYCOSYLASE7 (XTR7), was increased in the VQ29-OE plants under
far-red light condition (Figure 2F). Taken together, these results demonstrate that
VQ29 is a negative regulator of seedling de-etiolation under far-red and low white
light conditions.
Subcellular localization of VQ29
To determine the subcellular localization of VQ29, we first fused VQ29 with green
fluorescence protein (GFP) and transiently expressed this construct in Arabidopsis
protoplasts. The VQ29-GFP fusion protein was detected with multiple bands using the
GFP antibody, likely due to partial degradation of the fusion protein in vitro
(Supplemental Figure S2). Confocal microscopy revealed that the VQ29-GFP fusion
was likely localized to the nucleus, cytoplasm and plasma membrane (Figure 3A).
Furthermore, we generated stable transgenic plants expressing a translational fusion
of
VQ29
with
GFP
under
the
control
of
the
CaMV
35S
promoter
(Pro35S:VQ29-GFP). GFP fluorescence was distributed mainly in the nucleus of
hypocotyl cells from these transgenic plants (Figure 3B). To substantiate the
localization of VQ29, we isolated protein fractions from nucleus, cytoplasm and
plasma membrane of Pro35S:Myc-VQ29 (line #10) transgenic plants. As shown in
Figure 3C, Myc-VQ29 fusion protein was only detected in cell fractions isolated from
the nucleus, but not from plasma membrane or cytosol. Its localization in the
cytoplasm and plasma membrane in the protoplasts in Figure 3A might be caused by
over-expression of VQ29-GFP in the protoplasts.
Expression pattern of VQ29
To examine VQ29 expression in different tissues, we analyzed its transcript levels
using RT-PCR. Relatively high levels of VQ29 transcript were observed in the stem,
11
whereas expression was low in the root, rosette leaf, flower, and silique (Figure 4A).
To further visualize the expression pattern of VQ29, we generated transgenic plants
expressing the β–glucuronidase (GUS) reporter gene under the control of the VQ29
promoter sequence (2.0 kb upstream of the ATG translational start codon).
Histochemical staining showed that the GUS reporter gene was expressed in the
radicle, hypocotyl, stem, leaf vein, flower, and silique base, indicating that VQ29 may
function in these tissues (Figure 4B).
We then asked whether VQ29 is regulated by light. phyA, phyB, and cry1
photoreceptor mutants, along with the Col wild type, were grown in far-red, red, and
blue light conditions, respectively, for 5 d. As shown in Figure 4C, quantitative
RT-PCR assays showed that VQ29 expression was repressed by various light
treatments. Furthermore, the VQ29 transcript levels were up-regulated in the
phyA-211 and phyB-9 mutants under far-red and red light conditions compared with
the wild type, respectively. No pronounced difference in VQ29 expression was found
between the cry1 mutant and the wild type. Surprisingly, in the dark, VQ29 expression
was increased in phyB mutant, but was not affected by phyA and cry1 mutations,
suggesting that phyB plays a role in regulating VQ29 in the etiolated seedlings (Figure
4C). These observations suggest that light inhibits VQ29 expression in a
phytochrome-dependent
manner,
consistent
with
its
role
in
regulating
photomorphogenesis under far-red and low intensity light conditions. However, an
immunoblotting assay using the MYC antibody and the VQ29-OE plants showed that
the protein level of VQ29 was not regulated by light (Supplemental Figure S5).
VQ29 physically interacts with PIF1
Previous studies reported that VQ proteins could interact with various WRKY TFs
(Hu et al., 2013, Lai et al., 2011, Wang et al., 2010). The findings that VQ29
possesses transcriptional activity and is involved in regulating photomorphogenesis
prompted us to hypothesize that VQ29 activity might depend on its interaction with
other TF(s). Since PIF proteins, including PIF1, 3, 4 and 5, are bHLH-type TFs that
play a critical role in repressing photomorphogenesis (Leivar et al., 2008, Shin et al.,
12
2009), we tested their possible interaction with VQ29 in a yeast two-hybrid system.
VQ29 was fused with the LexA DNA-binding domain (LexA-VQ29) and various PIF
proteins were ligated with the activation domain of B42 (AD-PIFs). Co-expression of
LexA-VQ29 with AD-PIF1 or AD-PIF3 caused strong activation of the LacZ reporter
(Figure 5A, B), indicating that VQ29 interacts with PIF1 and PIF3 in yeast cells.
However, only mild interaction was detected between VQ29 and PIF5. As negative
controls, AD-SIG1 (plastid-located SIGMA FACTOR 1) or AD alone failed to interact
with LexA-VQ29 (Figure 5A, B). In this study, the relationship between VQ29 and
PIF1 was further analyzed.
To substantiate the interaction between VQ29 and PIF1 in plant cells, we
performed a bimolecular fluorescence complementation (BiFC) assay in which we
transiently co-expressed the N-terminus of yellow fluorescence protein fused to VQ29
(YFPN-VQ29) and PIF1 fused to the C-terminus of YFP (PIF1-YFPC) in Arabidopsis
protoplasts (Walter et al., 2004). Co-expression of YFPN-VQ29 and PIF1-YFPC
reconstituted a functional YFP in the nucleus, whereas co-expression with either
control vector failed to generate YFP fluorescence (Figure 5C). The in vivo
interaction
between
VQ29
and
PIF1
was
further
confirmed
by
co-immunoprecipitation assay. Therefore we generated double transgenic plants
Pro35S:VQ29-GFP/ Pro35S:TAP-PIF1 by crossing their single transgenic plant.
TAP-PIF1 (using MYC antibody) was able to pull down VQ29-GFP (detected by GFP
antibody) in the double transgenic seedlings (Figure 5D). In addition, two point
mutations in the VQ motif of VQ29 (V70D, Q71L) did not affect its interaction with
PIF1 (Figure 5C). These data together indicate that VQ29 indeed interacts with PIF1
and that the VQ motif is likely not required for mediating the interaction.
VQ29 and PIF1 co-regulate seedling de-etiolation
The physical interaction between VQ29 and PIF1 led us to investigate whether and
how these proteins function together in the light signaling pathway in Arabidopsis. To
this end, we generated the vq29 pif1 double mutant by crossing vq29-1 and pif1-2
mutants,
and
VQ29-OE
PIF1-OE
double
13
transgenic
plants
by
crossing
Pro35S:Myc-VQ29 (line #10) and Pro35S:TAP-PIF1. We examined the hypocotyl
elongation phenotypes of the double homozygous lines, their single mutant or
transgenic parent plants, and the wild type. The hypocotyl length of the vq29 pif1
double mutant was shorter than that of the single mutants and Col wild-type seedlings
under both far-red and low light conditions (Figure 6A, B, Supplemental Figure S3).
Furthermore, the vq29 pif1 double mutant was pronouncedly shorter than the single
mutants under far-red/dark cycles, or under far-red light conditions without sucrose
supplement in the media (Figure 6C, Supplemental Figure S4B). By contrast, the
VQ29-OE PIF1-OE double transgenic plants displayed much longer hypocotyls than
their parent single overexpression lines and the wild-type plants under low light
conditions (Figure 6D). These data together indicate that VQ29 and PIF1 additively
repress photomorphogenesis.
Since PIF1 is also involved in regulating phytochrome-mediated seed
germination and seedling growth during de-etiolation (Oh et al., 2004; Leivar et al.,
2008; Huq et al., 2004,), we next tested whether mutation or overexpression of VQ29
have these responses. As previously reported, pif1 mutant displayed high germination
rate after 5 min of far-red light treatment (Supplemental Figure S6A). Seeds of vq29
mutant and VQ29-OE did not show any germination difference compared to wild type,
and the germination efficiency of vq29 pif1 double mutant was similar as pif1
(Supplemental Figure S6A). In addition, mutation or overexpression of VQ29 did not
show seedling greening phenotype, and vq29-1 did not affect the defects of pif1
mutant during de-etiolation (Supplemental Figure S6B). These observations indicate
that VQ29 is not involved in seed germination and seedling greening responses with
PIF1, and that its responsiveness in hypocotyl elongation appears to be specific.
VQ29 binds to the promoters of PIL1 and XTR7 and regulates their expression
with PIF1
PIF1 also regulates the expression of light-responsive and cell elongation-related
genes, such as PIL1 and XTR7 (Leivar et al., 2012). Quantitative RT-PCR analysis
revealed that the expression of PIL1 and XTR7 was greatly down-regulated in
14
darkness in the vq29 pif1 double mutant compared with its parent mutants and the
wild type (Figure 7A). In addition, the transcript levels of another two genes involved
in cell elongation, EXTENSIN 1 (EXT1) and EXT3, were decreased in vq29 and pif1
mutants, and further reduced in the vq29 pif1 double mutant (Supplemental Figure S7).
We also tested the expression of EXPANSIN8 (EXP8) and EXP10, two genes that were
repressed by PIF1 (Oh et al., 2009). Surprisingly the EXP10 transcript level was
increased in vq29 pif1 double mutant compared that in pif1 (Supplemental Figure S7).
Furthermore, vectors harboring the LUC reporter gene driven either by the PIL1
or XTR7 promoter were constructed and co-transformed with VQ29 and/or PIF1 into
Arabidopsis protoplasts. As shown in Figure 7B, VQ29 itself did not significantly
promote LUC expression under the control of either the PIL1 or XTR7 promoter,
whereas PIF1 alone strongly activated LUC expression. Most remarkably,
co-expression of PIF1 and VQ29 increased the expression levels of these LUC
reporter genes. Interestingly, a mutated version of VQ29 (V70D) further activated the
levels of LUC expression when co-transformed with PIF1 (Figure 7B), suggesting that
the VQ domain of VQ29 indeed possesses transcription repression function.
Consistent with these observations, the expression of PIL1 and XTR7 was increased in
PIF1-OE transgenic plants, and XTR7’s level was further activated in VQ29-OE
PIF1-OE double transgenic plants (Figure 7C). Therefore, PIF1 and VQ29
coordinately regulate downstream gene expression, and VQ29 likely stimulates the
activation activity of PIF1.
To assess whether the induction of PIL1 and XTR7 is directly affected by PIF1
and VQ29, we carried out chromatin immunoprecipitation followed by quantitative
PCR (ChIP-qPCR) assays. When precipitated with GFP antibodies, fragments of the
PIL1 and XTR7 promoters containing the G-box (P), but not their coding regions (C),
were greatly enriched in extracts from Pro35S:VQ29-GFP transgenic seedlings, but
not from Col wild-type seedlings (Figure 7D, E). In addition, MYC antibody was able
to pull down the promoter fragments of XTR7, but not of PIL1, in extracts from
Pro35S:TAP-PIF1 transgenic plants (Figure 7F). These data indicate that VQ29 is
associated with the promoter regions of PIL1 and XTR7 in plant cells.
15
PIF1 is rapidly degraded after light transition (Shen et al., 2005; 2008). To test
whether VQ29 could affect the stability of PIF1, we performed an immunoblotting
assay using the MYC antibody, and PIF1-OE and PIF1-OE VQ29-OE transgenic
plants. Our result showed that overexpressing VQ29 did not affect the stability and
degradation of PIF1 (Supplemental Figure S8).
16
DISCUSSION
VQ29 defines a novel repressor of seedling de-etiolation
In this study, we present multiple lines of evidence that VQ29 is a novel transcription
regulator that represses seedling de-etiolation in Arabidopsis. First, hypocotyl
elongation in plants overexpressing VQ29 exhibits reduced sensitivity specifically to
far-red and low light conditions (Figure 2). It should be noted that although no effect
of vq29-1 mutant in far-red light was observed in Figures 2E and 6B, a statistically
significant short hypocotyl phenotype was observed in this mutant in low far-red light
intensities (Supplemental Figure S3) or in far-red light without sucrose, in multiple
independent experiments (Supplemental Figure S4). These opposite phenotypes of the
loss-of-function mutant and overexpression plants of VQ29 suggest that its coding
protein is involved in the phytochrome signaling pathway. In agreement with this,
VQ29 expression is down-regulated by light in a phytochrome-dependent manner
(Figure 4C). Second, yeast two-hybrid, BiFC and Co-IP assays showed that VQ29 is
able to physically interact with PIF1, a key transcription factor in the light signaling
pathway, both in yeast and in plant cells (Figure 5). Third, the facts that the vq29 pif1
double mutant or VQ29-OE PIF1-OE double transgenic plants exhibited pronounced
phenotypes compared with their corresponding single parent plants suggest that VQ29
and PIF1 have additive effect (Figure 6, Supplemental Figure S3, S4). Fourth, VQ29
and PIF1 function together to activate the expression of four genes involved in
promoting cell elongation. Furthermore, this regulation might be direct, as VQ29 is
associated with the promoters of PIL1 and XTR7 in vivo (Figure 7E). The less strong
phenotype in the vq29 mutant could be alternatively caused by the functional
redundancy of VQ29 with other related VQ genes. Future studies using double or high
order mutants with other VQs should further test this possibility.
PIF1 is stabilized in darkness and degraded by light (Shen et al., 2005, 2008). It
directly binds to DNA via the G-box cis-element in the promoter of downstream
genes and regulates their expression (Leivar and Quail, 2010). Consistently, a G-box
motif is found in the promoter regions of both PIL1 and XTR7. Therefore, we propose
17
that VQ29 transcript accumulates in darkness and its coding protein acts as a
transcription co-regulator to promote the activity of PIF1 through physical interaction,
leading to the activation of cell elongation-related genes (e.g., PIL1 and XTR7),
thereby inhibiting seedling de-etiolation (Figure 8). In far-red light conditions, the
levels of PIF1 protein and VQ29 transcript are reduced, resulting in the inhibition of
cell elongation-related gene expression, and consequently the promotion of
photomorphogenesis. Alternatively, VQ29 might function as a transcription factor that
directly binds to the promoter of downstream genes independent of PIF1. Further
study is deserved to examine this possibility.
PIL1 (encodes a PIF-regulated transcription factor) and XTR7 (encodes a
xyloglucan endotransglycosylase-related protein) are early shade marker genes
(Hornitschek et al., 2009) that are directly regulated by PIF1 and VQ29. VQ29 is
required for regulating photomorphogenesis specifically in low white light and far-red
light, which mimic the shade conditions. Hence, the involvement of VQ29 might fine
tune the responsiveness to shade-like environment by coordinating with PIF1.
Consistently, PIF1 also plays a role, although moderate, in shade avoidance response
(Leivar et al., 2012). Our ChIP experiment showed that VQ29 is associated with the
promoter region of XTR7. However, no difference in the expression of XTR7 in
vq29-1 mutant and wild type was found, suggesting that other factor(s) is required for
regulating downstream gene expression with VQ29. In agreement with this notion,
XTR7 mRNA level was greatly reduced in the vq29 pif1 double mutant. In addition, it
is also possible that other VQ member(s) plays a role in regulating downstream gene
expression with VQ29.
Accumulating studies suggest that members of the VQ family play diverse
functions, such as regulating the plant defense response (Narusaka et al., 2008, Xie et
al., 2010, Lai et al., 2011, Andereasson et al., 2005), abiotic stress resistance (Perruc
et al., 2004, Hu et al., 2013), and seed development (Wang et al., 2010). Our study
provides genetic and molecular evidence that VQ29 has a prominent role in plant
growth and development in response to the light environment, thus extending the
functionality of this plant-specific protein family.
18
VQ proteins are involved in transcriptional regulation
VQs are small proteins with a short, unique VQ motif. Molecular studies indicate that
VQ proteins likely interact with WRKY transcription factors and other proteins, such
as kinases and plastid sigma factors (Lai et al., 2011, Wang et al., 2010, Cheng et al.,
2012, Hu et al., 2013, Andereasson et al., 2005). WRKY and PIF are plant-specific
transcription factors that play crucial biological roles (Rushton et al., 2010, Leivar and
Quail, 2010). The interaction of VQ transcription regulators with other transcription
factors appears to fine-tune the temporal and spatial expression patterns of specific
targets during plant development, or upon exposure to particular biotic and abiotic
stresses. Our data showed that mutations in the VQ domain of VQ29 do not affect the
interaction with PIF1 (Figure 5C). However, a recent study revealed that the VQ motif
of SIB1 is important for its interaction with WRKY33 (Lai et al., 2011). Therefore,
there is variation in which domains of VQ proteins interact with other proteins.
VQ29 possesses transcriptional repression activity in a transient expression
system. Our detail analysis of the conserved V and Q residues in the VQ motif
indicates that point mutations of VQ29 alter its transcriptional activity (Figure 1D).
Moreover, mutation in VQ29 (V70D) further activates ProPIL1:LUC and
ProXTR7:LUC reporter expression mediated by PIF1 (Figure 7B). These observations
demonstrate that the VQ domain of VQ29 indeed has repressive function on gene
expression. However, VQ29 helps PIF1 to promote PIF1-induced gene expression,
and to repress the expression of PIF1-inhibited genes during light-mediated early
seedling growth. Hence, the activity of VQ29 and its effect on gene expression likely
depend on specific interacting factor and the target genes in a given signaling process.
Similarly, SIB1 simulates the DNA-binding and transcriptional activity of WRKY33
in the plant defense response (Lai et al., 2011). In contrast, recruitment of VQ9 leads
to the inactivation of WRKY8, and these proteins act antagonistically to mediate the
salt stress response (Hu et al., 2013).
Our transient expression assay in protoplasts suggests that the majority of VQ
proteins exhibit either transcriptional activation or repression activities (Figure 1).
19
The fact that VQ29 localizes to the nucleus further supports its role in regulating
transcription. Furthermore, almost all members of this family are thought to localize
to the nucleus, based on subcellular proteomic database predictions (Cheng et al.,
2012). The identification of more interacting proteins, particularly of transcription
factors in the nucleus, should further elucidate the functions and mechanisms of VQ
proteins in plants.
20
MATERIALS AND METHODS
Plant materials and growth conditions
The vq29-1 (Salk_061586 and Salk_061438), pif1-2, phyA-211, phyB-9 and cry1-304
mutants and Pro35S:TAP-PIF1 transgenic plants were derived from the Arabidopsis
thaliana Columbia (Col) ecotype (Chen et al., 2013, Reed et al., 1994, 1993, Mockler
et al., 1999, Moon et al., 2008). vq29-1 was confirmed by PCR genotyping and the
T-DNA insertion site was verified by sequencing. Double mutant or transgenic plants
were generated by genetic crossing and homozygous lines were used in these
experiments. Seedlings were grown on Murashige and Skoog medium containing 1%
sucrose, or without sucrose as indicated in the text and Supplemental Figure S4.
Far-red light (12 µmol m-2 s-1), red light (20 µmol m-2 s-1), and blue light (14 µmol m-2
s-1) were supplied by light-emitting diode light sources, and low white light (10 µmol
m-2 s-1) was supplied by cool white fluorescent lamps. For fluence rate analysis, the
light intensities were indicated in the figures.
Determination of Hypocotyl Length, Seedling Greening and Seed Germination
Rate
For hypocotyl length measurement, seedlings of different genotypes were grown side
by side on the same plate, and at least three independent plates were used in all
experiments. Seedlings were then photographed and their hypocotyl length was
measured by using Image J software (http://rsb.info.nih.gov/ij). For seedling greening
rate analysis, dark-grown seedlings were transferred to continuous white light for 2 d.
Greening rate was determined by counting the percentage of dark-green cotyledons
from 50 to 80 seedlings of each genotype. For seed germination assay, seeds of
different genotypes were harvested on the same day from plants grown in identical
conditions. Seed germination was observed under a microscope and determined based
on the appearance of radicle protrusion.
Gene expression analysis
21
Plant total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen), and first
strand cDNA was synthesized using Reverse Transcriptase (Invitrogen). Real-time
PCR was carried out using the SYBR Premix ExTaq Kit (Takara) following the
manufacturer’s instructions. The expression levels were normalized to the expression
of a ubiquitin (UBQ1) gene. Primers are listed in Supplemental Table S1.
Plasmid construction
To obtain open reading frames (ORFs) of VQ2, PIF4, and PIF5, first strand cDNA
was reverse transcribed from total RNA extracted from Col wild-type seedlings using
oligo (dT)18 primer and high fidelity Pfu DNA polymerase (Invitrogen). The products
were cloned into the pEASY vector (TransGen), resulting in pEASY-VQ2,
pEASY-PIF4, and pEASY-PIF5, respectively. Due to the absence of introns in all VQ
genes (except VQ2), the ORFs were amplified from genomic DNA using Pfu and
cloned into the pEASY vector, resulting in pEASY-VQs, respectively. Mutations in
VQ29 (V70A, V70D, Q71L, V70D Q71L) were generated using a MutantBEST
site-directed mutagenesis kit (Takara) according to the manufacture’s instructions.
Appropriate restriction enzyme sites were designed at the end of each primer
(Supplemental Table S1). All amplified ORFs were validated by sequencing.
To generate constructs for the protoplast transient expression assay, the VQ1
fragment was released from pEASY-VQ1 digested with BamHI and XhoI, and
inserted into the BglII/SalI site of pSAT-GAL4DB (Jing et al., 2013), to yield
pGAL4DB-VQ1. The ORFs of VQ4, VQ5, VQ6, VQ7, and VQ18 were released from
the corresponding MfeI-XhoI-digested pEASY-VQ vectors, respectively, and cloned
into the EcoRI/SalI sites of pSAT-GAL4DB, giving rise to pGAL4DB-VQ4/5/6/7/18.
The ORFs of VQ24 and VQ30 were released from pEASY-VQ24/30 digested with
EcoRI and SalI, and inserted into the EcoRI/SalI sites of pSAT-GAL4DB, generating
pGAL4DB-VQ24 and pGAL4DB-VQ30, respectively. To obtain pGAL4DB-VQ34,
the pEASY-VQ34 plasmid was cut with MfeI and SalI, and the VQ34 ORF was
cloned
into
the
EcoRI/SalI
sites
of
pSAT-GAL4DB.
The
ORFs
VQ2/3/8/9/10/11/12/13/14/15/16/17/19/20/21/22/23/25/26/27/28/29/31/32/33
22
of
were
released from the corresponding EcoRI-XhoI-digested pEASY-VQ vectors,
respectively, and inserted into the EcoRI/SalI sites of pSAT-GAL4DB, resulting in
the corresponding pGAL4DB-VQs constructs.
To construct vectors for the yeast two-hybrid assay, the pEASY-VQ29 plasmid
was cut with EcoRI and XhoI, and the VQ29 fragment was ligated into
EcoRI/XhoI-digested EG202 vector (Clontech), to give rise to pLexA-VQ29. The
pAD-PIF1 and pAD-PIF3 plasmids were constructed in a previous study (Chen et al.,
2013). The pEASY-PIF4 and pEASY-PIF5 plasmids were digested with EcoRI and
SalI, and the PIF4 or PIF5 fragments were inserted into the EcoRI/XhoI sites of the
JG4-5 vector (Clontech), resulting in pAD-PIF4 or pAD-PIF5, respectively.
To prepare constructs for the BiFC assay, the VQ29 fragment released from
EcoRI/XhoI–digested pEASY-VQ29 was inserted into the pUC-SPYNE vector
(Walter et al., 2004) digested with EcoRI and XhoI, to generate pYFPN-VQ29.
pYFPC-PIF1 was described in a previous study (Chen et al., 2013).
To study the subcellular localization of VQ29, the fragment released from
pEASY-VQ29 using EcoRI and XhoI was ligated into the EcoRI/SalI sites of the
modified pdGN vector (Lee et al., 2005), to generate pGFP-VQ29.
To construct ProVQ29:GUS, a fragment spanning the region 2-kb upstream of the
ATG start site of VQ29 was amplified by PCR and cloned into pEASY, resulting in
pEASY-VQ29p. The pEASY-VQ29p plasmid was then digested with SalI and BamHI
to release the VQ29 promoter fragment, which was then ligated into the pBI101 vector
digested with the same enzymes, to generate ProVQ29:GUS.
To construct VQ29 overexpression vectors, the VQ29 coding sequence was
released from pEASY-VQ29 using EcoRI and XhoI, and then ligated into the
EcoRI-SalI sites of modified pRI101 (Takara), in which three copies of MYC tag
were inserted, resulting in Pro35S:Myc-VQ29. The VQ29 ORF was amplified from
pEASY-VQ29 by introducing an NcoI site at the N-terminal and a SpeI site at the
C-terminal,
and
cloned
into
pEASY
to
produce
pEASY-VQ29G.
The
pEASY-VQ29G plasmid was then digested with NcoI and SpeI and the VQ29
fragment was inserted into pCAMBIA1302 (www.cambia.org/daisy/cambia/585) cut
23
with the same enzymes, to generate Pro35S:VQ29-GFP.
To generate constructs for the transient expression of PIL1 and XTR7, the
promoter sequences of both genes were amplified from Col genomic DNA and ligated
into the pEASY vector, resulting in pEASY-PIL1p and pEASY-XTR7p, respectively.
These plasmids were cut with HindIII and BamHI to release the PIL1 and XTR7
fragments, which were inserted into the HindIII/BamHI sites of the pUC-35sLUC
vector (Chen et al., 2013) to produce ProPIL1:LUC and ProXTR7:LUC, respectively.
The binary constructs were electroporated into Agrobacterium tumefaciens strain
GV3101 and then introduced into Col wild type plants. Transgenic plants were
selected on MS plates in the presence of 50 mg/L kanamycin or hygromycin, and
homozygous lines were used in this study.
Luciferase transient expression assay
The LUC transient expression assay was carried out as previously described (Jing et
al., 2013). LUC reporter activity was detected with a luminescence kit using
Luciferase Assay System (Promega) and the relative activity was expressed as the
LUC/GUS ratios.
GUS histochemical assay
Seedlings of the ProVQ29:GUS transgenic line were harvested and incubated
overnight in 0.1 M sodium phosphate buffer containing 50 mM K3Fe(CN)6, 50 mM
K4Fe(CN)6, and 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide at 37°C. GUS
expression was examined under a dissecting microscope and images were captured by
a digital camera (Olympus).
GFP protein localization assay
For the transient assay, VQ29-GFP was transformed into Arabidopsis protoplasts and
the protoplasts were incubated under darkness for 16 h before observation. For GFP
localization in stable transgenic plants, a homozygous line of Pro35S:VQ29-GFP was
used. The protoplasts and transgenic seedlings were mounted on a slide and GFP
24
fluorescence was visualized with a Leica TCS SP5 confocal microscope.
Yeast two-hybrid assay
Yeast two-hybrid analysis was performed as previously described (Tang et al., 2012).
Transformants were grown on SD/-Trp-Ura-His dropout plates containing X-gal
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for color development. Relative
beta-galactocidase activity was quantified according to the method described by Yeast
Protocols Handbook (Clontech).
Co-IP and Immunoblotting
Seedlings were homogenized in extraction buffer containing 50 mM Tris-HCl, pH7.5,
150mM NaCl, 10 mM MgCl2, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride,
and 1× complete protease inhibitor cocktail (Roche). The extracts were centrifuged at
14,000 ×g twice at 4°C for 10 min each, and protein concentration was determined
using the Bradford assay (Bio-Rad). Protein fractionations from nucleus, plasma
membrane or cytoplasm were isolated according to the methods as described (Larsson
et al., 1994, Liu et al., 2001). Co-IP assay was carried out as previously described
(Tang et al., 2012). Proteins were separated on 10% SDS-PAGE gels and transferred
onto polyvinylidene fluoride membranes. They were then blotted against anti-MYC
(Abcam), anti-GFP (Sigma), anti-H+-ATPase (Agrisera), anti-cFBPase (Agrisera), or
anti-tubulin (Jing et al., 2013) antibodies. The protein bands were visualized using the
standard enhanced chemiluminescence method.
BiFC
Plasmids containing N- and C-terminal YFP fusions were co-transformed into
Arabidopsis protoplasts as previously described (Walter et al., 2004). The protoplasts
were incubated under weak light for 12-16 h before observation. YFP fluorescence
was captured with a confocal microscope (Leica).
ChIP
25
The Col wild type and Pro35S:TAP-PIF1 and Pro35S:VQ29-GFP transgenic plants
were used in the ChIP assay, which was carried out as previously described (Chen et
al., 2013). The chromatin complexes were incubated with anti-MYC or anti-GFP
polyclonal antibodies. The precipitated DNA fragments were recovered and quantified
by quantitative PCR with the primers shown in Supplemental Table S1.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or
GenBank/EMBL databases under the following accession numbers: VQ29
(At4g37710), PIF1 (At2g20180), PIF3 (At1g09530), PIF4 (At2g43010), PIF5
(At3g59060), PIL1 (At2g46970), XTR7 (At4g14130), EXT1 (At1g76930), EXT3
(At1g21310), EXP8 (At2g40610), EXP10 (At1g26770), SIG1 (At1g08540), ACTIN
(At3g18780) and UBQ1 (At3g52590), and the accession numbers of all other VQ
genes are listed in Supplemental Table S1.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Comparison of VQ genes in Arabidopsis, rice, and moss.
Supplemental Figure S2. VQ29 protein level in wild type and various point mutants.
Supplemental Figure S3. Fluence-rate response under different light conditions.
Supplemental Figure S4. Hypocotyl length of VQ29 mutant and overexpression plants
grown in the media without sucrose.
Supplemental Figure S5. VQ29 protein level during dark-to-light transition.
Supplemental Figure S6. Phenotype in seedling greening and seed germination.
Supplemental Figure S7. Downstream gene expression in vq29 and/or pif1 mutants.
Supplemental Figure S8. The effect of VQ29 on PIF1 protein stability.
Supplemental Table S1. List of primers used in this study.
ACKNOWLEDGMENTS
26
We thank the Arabidopsis Biological Resource Center for providing the vq29 mutant.
We are grateful to Dr. Enamul Huq (University of Texas at Austin) for providing the
mutant and overexpression transgenic seeds of PIF1, and Dr. Tai Wang (Institute of
Botany, Chinese Academy of Sciences) for gifting plasma membrane- and
cytosol-localized antibodies.
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Figure Legends
Figure 1. VQ proteins possess transcriptional activity.
(A) Diagrams of constructs used in the transient expression assay.
(B, C) Relative luciferase reporter (LUC) activity by various VQ effectors.
(D) Relative LUC reporter activity by VQ29 and its point mutants. For (B-D), the
effectors, LUC reporter, and GUS internal control were co-transformed into
Arabidopsis protoplasts. Data denote the mean ± SD of three biological replicates.
Asterisks in (B and C) indicate significant difference from the empty vector at P <
0.05 (single asterisk) or 0.01 (double asterisks) using Student’s t-test. Asterisks in (D)
indicate significant difference from the wild-type VQ29 at P < 0.01 (double asterisks)
using Student’s t-test.
Figure 2. Phenotypic analysis of the VQ29 loss-of-function mutant and
overexpression plants.
(A) Diagram of VQ29 gene structure and the position of the T-DNA insertion. Black
rectangle, exon of VQ29. Triangle, T-DNA insertion.
(B) Detection of VQ29 transcript level in the wild type and vq29-1 mutant by
real-time RT-PCR. Seedlings were grown in white light conditions (10 µmol m-2 s-1)
for 5 d. Data represent the mean ± SD of biological triplicates.
(C) Immunoblot analysis of two Pro35S:Myc-VQ29 overexpression transgenic lines
(VQ29-OE, line #10 and #14) and the wild type using anti-MYC antibody.
Immunoblotting against the tubulin antibody (anti-TUB) served as a loading control.
Seedlings were grown in low white light (10 µmol m-2 s-1) for 5 d before harvesting.
(D) Photomorphogenic responses under far-red (12 µmol m-2 s-1), low white light (10
µmol m-2 s-1) or in darkness. Seedlings were grown in various conditions for 5 d. Bars
indicate 5 mm.
(E) Quantification of hypocotyl length of the seedlings shown in (D). Data are mean ±
SD of at least 20 seedlings. Asterisks indicate significant difference from the wild
type at P < 0.05 (single asterisk) or 0.01 (double asterisks) using Student’s t-test.
33
(F) Expression of PIL1 and XTR7 by qRT-PCR. Four-day-old dark-grown seedlings
were transferred to far-red light for 12 h. Error bars indicate the SD of biological
triplicates.
Figure 3. Localization of VQ29.
(A) The VQ29-GFP and PIF1-GFP constructs and empty vector controls were
transformed into Arabidopsis protoplasts. Chlorophyll auto-fluorescence was shown
in red. Representative images are shown. Bar denotes 5 µm.
(B) GFP fluorescence in the hypocotyl of Pro35S:VQ29-GFP transgenic seedling
coincides with DAPI staining (which marks nucleus). Seedlings were grown in
darkness for 4 d. Bar denotes 50 µm.
(C) Immunoblotting assay of VQ29. The Pro35S:Myc-VQ29 (line #10) transgenic
plants were grown in 16 h light / 8 h dark conditions for 3 weeks. Immunoblotting
with antibodies against cytoplasm-localized fructose-1,6-bisphosphatase (cFBPase)
and plasma membrane-targeted H+-ATPase (Iwata et al., 2008) serves as positive
controls. Asterisk indicates the specific band. T, total protein; PM, plasma membrane;
C, cytoplasm; N, nucleus.
Figure 4. Expression pattern of VQ29.
(A) RT-PCR of VQ29 in various tissues. Amplified actin served as loading controls.
(B) GUS staining in various tissues of ProVQ29:GUS transgenic plants.
(C) Quantitative RT-PCR analysis of VQ29 in the photoreceptor mutants under
various light conditions and darkness. Seedlings were grown in the indicated
conditions for 5 d. Data indicate ± SD of three biological triplicates.
Figure 5. VQ29 physically interacts with PIF1.
(A) Yeast two-hybrid analysis of the interaction between LexA-VQ29 (fused with the
LexA DNA-binding domain) and various proteins tagged with the B42 activation
domain (AD). “-” means empty AD vector.
(B) Relative beta-galactocidase activity of interactions between LexA-VQ29 with
34
AD-tagged proteins shown in (A). Data indicate ± SD of six individual yeast colonies.
Asterisks indicate significant difference from the combination of LexA-VQ29 and AD
at P < 0.01 using Student’s t-test.
(C) BiFC assay showing the interaction between PIF1-YFPC and YFPN-VQ29 or
YFPN-VQ29(V70D,Q71L). Chlorophyll auto-fluorescence was shown in red. Bar
denotes 5 µm.
(D) Co-immunoprecipitation assay between VQ29 and PIF1. Pro35S:VQ29-GFP and
Pro35S:VQ29-GFP / Pro35S:TAP-PIF1 seedlings were grown in darkness for 5 d.
After precipitation with anti-MYC antibody (TAP-PIF1 contains MYC tag), proteins
were immunoblotted with anti-MYC or anti-GFP antibodies. IP, immunoprecipitation.
Figure 6. Analysis of double mutants and overexpression plants of VQ29 and
PIF1.
(A) Photomorphogenic phenotype of the Col wild type, vq29, pif1, and vq29 pif1
mutants in darkness, or under far-red (12 µmol m-2 s-1) or low white light (10 µmol
m-2 s-1) conditions. Bars denote 5 mm.
(B) Hypocotyl length of seedlings shown in (A). Seedlings were grown in the
indicated light conditions for 5 d.
(C) Hypocotyl length of seedlings grown under far-red-dark photocycles.
Three-day-old dark-grown seedlings were transferred to far-red-dark (FR 12h / D 12h)
condition at the beginning of light treatment and grown for an additional 2 d.
(D)
Hypocotyl
length
of
Pro35S:Myc-VQ29
(VQ29-OE,
line
#10)
and
Pro35S:TAP-PIF1 (PIF1-OE) plants and their corresponding double transgenic line
under darkness or low light conditions (10 µmol m-2 s-1). Seedlings were grown in the
indicated light conditions for 5 d. In (B-D), data are mean ± SD of at least 20
seedlings. Asterisks indicate significant difference from the wild type at P < 0.05
(single) or 0.01 (double) using Student’s t-test.
Figure 7. VQ29 and PIF1 co-regulate downstream gene expression.
(A) Relative expression of PIL1 and XTR7 by qRT-PCR. Seedlings were grown in
35
darkness for 4 d and then kept in darkness or transferred to far-red light (12 µmol m-2
s-1) for an additional 1 h. Error bars indicate ± SD of triplicates.
(B) Relative luciferase (LUC) reporter activity in Arabidopsis protoplasts
co-transformed with the effector constructs. LUC gene was under the control of either
PIL1 or XTR7 promoter. Data represent the mean ± SD of triplicates. Asterisks
indicate significant difference at P < 0.01 using Student’s t-test.
(C) Relative expression of PIL1 and XTR7 in VQ29 and/or PIF1 overexpression
plants by qRT-PCR. Seedlings were grown in darkness for 5 d. Error bars indicate ±
SD of triplicates.
(D) Diagram of the genomic structures of PIL1 and XTR7. Rectangles denote exons
and triangles represent G-box motifs. Amplicons used in the ChIP assay are
underlined. bp, base pairs.
(E, F) ChIP-qPCR assay showing the relative enrichment of fragments corresponding
to the promoters or coding regions of PIL1, XTR7, and the UBQ1 control. DNA was
precipitated with anti-GFP antibody in Pro35S:VQ29-GFP (E), or anti-MYC antibody
in Pro35S:TAP-PIF1 (F) transgenic plants, respectively. Data represent the mean ±
SD of triplicates.
Figure 8. A proposed model of VQ29 in regulating seedling de-etiolation.
Far-red light, which is perceived by phytochromes, represses PIF1 and VQ29 levels.
VQ29 interacts with PIF1 and acts as a transcriptional co-regulator to promote PIF1’s
activation activity on cell elongation-related genes, leading to the suppression of
seedling de-etiolation. Meanwhile, VQ29 might regulate downstream gene expression
independent of PIF1. Arrows denote positive effect, and bars indicate negative role.
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